轴突生长抑制性蛋白

轴突生长抑制性蛋白是阻止成年哺乳动物中枢神经再生的重要因素.近年来一些重要的抑制性蛋白陆续得到鉴定和克隆.本文概述轴突生长抑制性蛋白的发现过程,着重介绍勿动蛋白(Nogo),髓磷脂相关性糖蛋白(MAG)以及崩溃蛋白(Collapsin)的结构和功能,同时介绍有关轴突生长抑制性蛋白的受体和信号转导等方面的研究进展.     

NgR1复合物的实验研究进展与干扰策略

成年哺乳动物周围神经系统损伤后可有效再生,但中枢神经系统损伤后却很难再生。在分子途径促进损伤中枢神经系统轴突再生的研究中,发现了3种髓磷脂相关抑制性蛋白：Nogo、髓鞘相关糖蛋白（myelinassociatedglycoprotein,MAG）、少突胶质细胞髓鞘糖蛋白（oligodendroeytemyelinglycoprotein,OMgp）在中枢神经系统损伤后发挥着抑制轴突生长的作用,并发现Nogo—A、MAG、OMgp存在于中枢白质的髓鞘内外环和少突胶质细胞的表面,通过与共同受体NgR1特异性结合诱导生长锥塌陷并抑制轴突生长。进而提出了通过阻断NgR1复合物及其下游的信号转导途径来促进神经元轴突再生的设想。本文拟对近年来关于NgR1复合物的研究加以综述。     

S100β与Alzheimer病

Ｓ１００β是一种钙结合蛋白,在脑内它是轴突生长因子。正常情况下发挥重要的生理功能,但含量过高则具有神经毒性。本文综述了Ｓ１００β在Ａｌｚｈｅｉｍｅｒ病（ＡＤ）各种可能的发病机制中的介导作用,尤其是它与炎症介质及β－淀粉样蛋白（Ａβ）三者之间相互作用,参与轴突变性和老年斑的形成,促成了ＡＤ的重要病理变化,值得高度重视。进一步探明过高水平的Ｓ１００β对神经元的影响,将有助于阐明ＡＤ的致病机制。     

RhoA与神经轴突的生长

成年哺乳动物中枢神经系统（CNS）轴突损伤后很难再生的一个重要原因是损害局部环境中存在一些生长抑制因子，目前发现轴突生长抑制因子Nogo—A，髓鞘相关糖蛋白（MAG）和少突胶质细胞髓鞘糖蛋白（OMGP）均通过相同的受体NgR介导，在p75的参与下，影响细胞内RhoA信号通路抑制神经轴突再生。现将RhoA与神经轴突生长研究进展进行简要综述。     

脑衰反应调节蛋白-2（CRMP-2）促进海马神经元轴突生长

目的研究脑衰反应调节蛋白2（collapsin response mediator protein,CRMP-2）对原代海马神经元轴突生长的影响,探讨CRMP-2在神经元轴突生成中的作用。方法每次原代取孕18d SD大鼠1只,每只孕鼠含胎鼠约12~15只。显微镜下分离12~15只胎鼠双侧海马组织,消化法培养原代海马神经元。原代海马神经元培养采用核电转实验转染绿色荧光蛋白（enhanced green fluorescence protein,EGFP）和野生型CRMP-2（wild type（wt）CRMP2）和突变型T514D-CRMP2（突变体,模拟失活型CRMP-2）。培养72h固定做免疫荧光双标,分别绿色荧光蛋白（EGFP）和轴突标志物Tau-1,采用激光共聚焦显微镜和普通光学显微镜观察海马神经元形态变化。结果转染EGFP载体的对照组神经元正常发育,培养至72h,神经元轴突特异性表达标志物蛋白Tau-1;过表达了wtCRMP-2的神经元除了生成一条长的特异性表达Tau-1的轴突,另外一条突起也发育成了特异性表达Tau-1的轴突;而过表达了失活型CRMP-2的神经元发育与正常组相比无差异。结论野生型CRMP-2可明显促进轴突生长,而转染模拟磷酸化CRMP-2的突变体T514D-CRMP2则没有显示任何的促进作用。     

细胞外信号调节激酶在中枢神经系统中的研究进展

细胞外信号调节激酶（extracellular signal-regulatedkinase．ERK）是丝裂原活化蛋白激酶（mitogen activated proteinkinase，MAPK）的家族成员之一，也是迄今为止哺乳动物细胞中研究最深入的MAPK成员。它在介导细胞增殖、分化及存活中发挥重要作用，可被神经递质、神经营养因子、生长因子等多种信号激活。在中枢神经系统中目前研究发现ERK可通过基因表达、蛋白合成等影响神经细胞增殖分化、突触可塑性、轴突生长等，并且参与多种神经系统疾病的病理生理过程。     

γ-氨基丁酸在中枢神经系统发育中的作用及机制

氨基丁酸（γ-Aminobutyricacid,GABA）是成年哺乳动物中枢神经系统内的抑制性神经递质。在中枢神经系统发育过程中,GABA由兴奋性神经递质转变为抑制性神经递质。其转变过程主要表现为GABA的释放由促进突触后神经元的Ca^2＋内流变为抑制突触后神经元的Ca^2＋内流。中枢神经元内GABA作用的转变受细胞内Cl^-浓度的影响：当细胞内Cl^-浓度处于高水平时GABA发挥兴奋性神经递质的作用,当细胞内Cl^-浓度降低到一定程度后GABA发挥抑制性神经递质的作用。升高中枢神经元内Cl^-浓度的是Na^＋-K^＋-Cl^-Cl^-同向转运蛋白1（Na^＋K^＋Cl^--Cl^-cotransporters1,NKCC1）,而K^＋-Cl^-协同转运蛋白2（K^＋-Cl^-cotransporter2,KCC2）则使中枢神经元内Cl^-浓度降低。     

NOGO—A在PC12细胞分化过程中的表达及意义

目的探讨NOGO—A在PC12细胞生长发育过程中的表达及意义。方法培养大鼠嗜铬细胞瘤细胞系PC12细胞，用神经生长因子诱导其分化，并于倒置显微镜下随机取20视野计数观察细胞增值和轴突生长情况。采用免疫荧光染色、逆转录酶聚合酶链反应（RT—PCR）及免疫印迹法等方法检测诱导后第1d、第3d、第5d、第7d PC12细胞中NOGO—AmRNA及蛋白的表达及变化，并留取细胞培养液检测多巴胺水平。结果未分化的PC12细胞中未检测到NOGO—A mRNA及蛋白表达。经神经生长因子诱导的PC12细胞，细胞轴突不断生长，NOGO—AmRNA及蛋白的表达逐渐增高（P〈0．05）。PC12细胞在分化过程中多巴胺（DA）分泌水平无明显差别。结论PC12细胞向交感神经元分化的过程中NOGO—A的表达逐渐增强，推测NOGO—A在神经元发育早期可能促进轴突生长。但对多巴胺激素释放的调节不明显。     

冈田酸抑制神经元轴突生长

目的研究蛋白磷酸酯酶2A（protein phosphatase2A，PP2A）抑制剂冈田酸（okadaic acid，OA）对胎鼠海马神经元轴突生长的影响，探讨PP2A在神经元轴突生成中的作用。方法实验分为二甲基亚砜（Dimethyl sulfoxide，DMSO）对照组和0A处理组，OA处理组依药物浓度又分为5、10、20、30、40nmol／L5个组，激光扫描共聚焦显微镜和普通光学显微镜观察海马神经元形态变化。结果对照组原代海马神经元培养48h后神经元轴突已经形成。当给予不同浓度的0A处理后普通光学显微镜下可见原代海马神经元突起生长均明显受到抑制，突起长度缩短随OA浓度增加抑制作用逐渐增强，对照组平均轴突长度为185pm，而不同浓度OA处理后则分别下降至150／μm、100,am、80ptm、75μm、50μm。结论OA抑制原代海马神经元轴突生长，提示PP2A在神经元轴突生成中起重要作用。     

microRNA在中枢神经系统神经再生中作用

正中枢神经系统神经损伤后再生困难主要由内在再生能力低下和外部抑制性环境所致。微小RNA(micro RNA,mi RNA)是神经元内调控轴突生长能力的重要因子,也可以通过调节外部再生抑制因子发挥作用。本文就mi RNA在中枢神经系统再生中作用进行综述。1 mi RNA与神经再生mi RNA是一类大小约22个碱基的内源性非编码单链RNA分子,参与基因转录后的表达调控~([1])。     

Novel roles for Nogo receptor in inflammation and disease

The Nogo receptor (NgR), which was identified as a common receptor for three axon growth inhibitors associated with myelin, has been extensively characterized for its role in triggering growth cone collapse and arresting neurite/axon growth. Recent studies indicate that NgR is also expressed in nonneuronal cells and modulates macrophage responses during inflammation after peripheral nerve injury. In this article, we discuss the possibility that NgR might have wider effects on inflammation in a variety of neurological conditions ranging from central nervous system trauma to diseases such as multiple sclerosis or Alzheimer's disease.     

Nogo, a star protein in reticulon family

Nogo is widely expressed in higher vertebrate animals. Nogo gene gives rise to multiple isoforms. All the subtypes of Nogo proteins are characterized by a 200-amino-acid C-terminal domain, including two long hydrophobic sequences. Biological functions of Nogo include inhibition of neurite growth from the cell surface via specific receptors, intracellular trafficking, cell division and apoptosis. Here, we briefly review the elementary structure, taxonomic distribution and tissue expression of Nogo, summarize recent discoveries about localization of Nogo and mechanism of action, and discuss the possible functions of Nogo.     

Nogo,a star protein in reticulon family

1 Introduction In higher vertebrate animals, from amphibian to mammalian, when the central nervous system (CNS) is injured, the neural tissue itself is difficult for most animals to regenerate. Earlier functional studies indicated that in- hibitory proteins, such as myelin-associated glycoprotein (MAG)[1], oligodendrocyte-myelin glycoprotein (OMgp)[2 ] and Nogo[3], are enriched in myelin and oligodendrocytes. Among these proteins, Nogo has attracted more and more interest, although the fun…     

Substrate properties of zebrafish Rtn4b/Nogo and axon regeneration in the zebrafish optic nerve

This study explored why lesioned retinal ganglion cell (RGC) axons regenerate successfully in the zebrafish optic nerve despite the presence of Rtn4b, the homologue of the rat neurite growth inhibitor RTN4‐A/Nogo‐A. Rat Nogo‐A and zebrafish Rtn4b possess characteristic motifs (M1‐4) in the Nogo‐A‐specific region, which contains delta20, the most inhibitory region of rat Nogo‐A. To determine whether zebrafish M1‐4 is inhibitory as rat M1‐4 and Nogo‐A delta20, proteins were recombinantly expressed and used as substrates for zebrafish single cell RGCs, mouse hippocampal neurons and goldfish, zebrafish and chick retinal explants. When offered as homogenous substrates, neurites of hippocampal neurons and of zebrafish single cell RGCs were inhibited by zebrafish M1‐4, rat M1‐4, and Nogo‐A delta20. Neurite length increased when zebrafish single cell RGCs were treated with receptor‐type‐specific antagonists and, respectively, with morpholinos (MO) against S1PR2 and S1PR5a—which represent candidate zebrafish Nogo‐A receptors. In a stripe assay, however, where M1‐4 lanes alternate with polylysine‐(Plys)‐only lanes, RGC axons from goldfish, zebrafish, and chick retinal explants avoided rat M1‐4 but freely crossed zebrafish M1‐4 lanes—suggesting that zebrafish M1‐4 is growth permissive and less inhibitory than rat M1‐4. Moreover, immunostainings and dot blots of optic nerve and myelin showed that expression of Rtn4b is very low in tissue and myelin at 3–5 days after lesion when axons regenerate. Thus, Rtn4b seems to represent no major obstacle for axon regeneration in vivo because it is less inhibitory for RGC axons from retina explants, and because of its low abundance.     

Nogos and the Nogo-66 receptor: factors inhibiting CNS neuron regeneration

The recently cloned gene Nogo, whose alternative splice products correspond to the antigenic target of the central nervous system (CNS) regeneration enhancing monoclonal antibody IN-1, codes for membrane proteins enriched in brain, particularly in oligodendrocytes. The 66-amino acid extracellular domain of Nogo (Nogo-66) interacts with a high-affinity receptor (NgR), a glycosylphosphatidylinositol (GPI)-linked protein with multiple leucine-rich repeats. The amino terminal cytoplasmic domain of Nogo appears to have a general cellular growth inhibitory effect. Nogo-66, on the other hand, specifically retards neurite outgrowth and induces growth cone collapse, possibly through its interaction with NgR and as yet unidentified transmembrane coreceptors. Recent results also suggest that Nogo expression may induce apoptosis in tumor cells. Together, these proteins provide new molecular handles for the design of therapeutic interventions for CNS injuries and neurodegenerative diseases, as well as possible leads to anticancer strategies.     

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Myelin-associated glycoprotein(MAG) inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor(NgR) and paired immunoglobulin-like receptor B(PirB) was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase(ROCK) phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.     

Mild hypothermia combined with a scaffold of NgR- silenced neural stem cells/Schwann cells to treat spinal cord injury

Because the inhibition of Nogo proteins can promote neurite growth and nerve cell differentiation, a cell-scaffold complex seeded with Nogo receptor(Ng R)-silenced neural stem cells and Schwann cells may be able to improve the microenvironment for spinal cord injury repair. Previous studies have found that mild hypothermia helps to attenuate secondary damage in the spinal cord and exerts a neuroprotective effect. Here, we constructed a cell-scaffold complex consisting of a poly(D,L-lactide-co-glycolic acid)(PLGA) scaffold seeded with Ng R-silenced neural stem cells and Schwann cells, and determined the effects of mild hypothermia combined with the cell-scaffold complexes on the spinal cord hemi-transection injury in the T9 segment in rats. Compared with the PLGA group and the Ng R-silencing cells + PLGA group, hindlimb motor function and nerve electrophysiological function were clearly improved, pathological changes in the injured spinal cord were attenuated, and the number of surviving cells and nerve fibers were increased in the group treated with the Ng R-silenced cell scaffold + mild hypothermia at 34°C for 6 hours. Furthermore, fewer pathological changes to the injured spinal cord and more surviving cells and nerve fibers were found after mild hypothermia therapy than in injuries not treated with mild hypothermia. These experimental results indicate that mild hypothermia combined with Ng R gene-silenced cells in a PLGA scaffold may be an effective therapy for treating spinal cord injury.     

Rho-ROCK inhibitors for the treatment of CNS injury

Injured axons in the adult central nervous system (CNS) exhibit almost no regeneration. Several myelin-associated proteins such as myelin-associated glycoprotein (MAG), Nogo, and oligodendrocyte-myelin glycoprotein (OMgp) have been identified as inhibitors of CNS axonal regeneration in the CNS. Recently, repulsive guidance molecule (RGM) was identified as a potential myelin-derived neurite outgrowth inhibitor in vitro and in vivo. These axonal growth inhibitors transmit inhibitory signals through common intracellular molecules such as RhoA and its effector Rho kinases (ROCK). The effects of these axonal growth inhibitors are blocked by inhibition of the Rho-ROCK pathway in vitro. Injuries to the adult CNS induce the activation of the Rho-ROCK pathway, and the inhibition of this pathway promotes axonal regeneration and functional recovery in the injured CNS. Therefore, the Rho-ROCK pathway is a promising target for drug development for the treatment of human CNS injuries such as spinal cord injuries. This review also discusses recent patents and future developments which are useful in the treatment of human CNS injuries.     

LOTUS suppresses axon growth inhibition by blocking interaction between Nogo receptor-1 and all four types of its ligand

Axon growth inhibitors such as Nogo proteins, myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp), and B lymphocyte stimulator (BLyS) commonly bind to Nogo receptor-1 (NgR1), leading to enormous restriction of functional recovery after damage to the adult central nervous system. Recently, we found that lateral olfactory tract usher substance (LOTUS) antagonizes NgR1-mediated Nogo signaling. However, whether LOTUS exerts antagonism of NgR1 when bound by the other three ligands has not been determined. Overexpression of LOTUS together with NgR1 in COS7 cells blocked the binding of MAG, OMgp, and BLyS to NgR1. In cultured dorsal root ganglion neurons in which endogenous LOTUS is only weakly expressed, overexpression of LOTUS suppressed growth cone collapse and neurite outgrowth inhibition induced by these three NgR1 ligands. LOTUS suppressed NgR1 ligand-induced growth cone collapse in cultured olfactory bulb neurons, which endogenously express LOTUS. Growth cone collapse was induced by NgR1 ligands in lotus-deficient mice. These data suggest that LOTUS functions as a potent endogenous antagonist for NgR1 when bound by all four known NgR1 ligands, raising the possibility that LOTUS may protect neurons from NgR1-mediated axonal growth inhibition and thereby may be useful for promoting neuronal regeneration as a potent inhibitor of NgR1.     

Upregulation of axon guidance molecules in the adult central nervous system of Nogo‐A knockout mice restricts neuronal growth and regeneration

Adult central nervous system axons show restricted growth and regeneration properties after injury. One of the underlying mechanisms is the activation of the Nogo‐A/Nogo receptor (NgR1) signaling pathway. Nogo‐A knockout (KO) mice show enhanced regenerative growth in vivo, even though it is less pronounced than after acute antibody‐mediated neutralization of Nogo‐A. Residual inhibition may involve a compensatory component. By mRNA expression profiling and immunoblots we show increased expression of several members of the Ephrin/Eph and Semaphorin/Plexin families of axon guidance molecules, e.g. EphrinA3 and EphA4, in the intact spinal cord of adult Nogo‐A KO vs. wild‐type (WT) mice. EphrinA3 inhibits neurite outgrowth of EphA4‐positive neurons in vitro. In addition, EphrinA3 KO myelin extracts are less growth‐inhibitory than WT but more than Nogo‐A KO myelin extracts. EphA4 KO cortical neurons show decreased growth inhibition on Nogo‐A KO myelin as compared with WT neurons, supporting increased EphA4‐mediated growth inhibition in Nogo‐A KO mice. Consistently, in vivo, Nogo‐A/EphA4 double KO mice show increased axonal sprouting and regeneration after spinal cord injury as compared with EphA4 KO mice. Our results reveal the upregulation of developmental axon guidance cues following constitutive Nogo‐A deletion, e.g. the EphrinA3/EphA4 ligand/receptor pair, and support their role in restricting neurite outgrowth in the absence of Nogo‐A.